The long-term objective or our work is to reconstruct the pathogenesis of human diabetic retinopathy at the cellular and molecular level, and thus establish the foundation for preventive strategies rationally targeted. We address in particular the process that leads to capillary closure and obliteration because these are the events that trigger sight-threatening unregulated angiogenesis. Having observed in both human and experimental diabetes accelerated apoptosis of the cells of retinal vessels - pericytes and endothelial cells - we sought to identify relevant molecular events. Two candidate pathways are emerging: one driven by intracellular regulators of apoptosis, and in particular by overexpression of proapoptotic Bax; and one by proinflammatory signals, and in particular by activation of the transcription factor NF-kappaB and overexpression of TNF-alpha. We propose to (1) define in retinal pericytes exposed to high glucose in vitro the respective apoptogenic role of Bax and TNF-alpha overexpression, their relationship to NF-kappaB activation, possible transcriptional regulation by high glucose, and relationship to by-products of accelerated glycolysis; (2) determine if endothelial cells derived from the same retinas as index pericytes respond to high glucose in vitro with the same proapoptotic program; (3) determine--by studying mice with targeted deletion of Bax and rendered diabetic--, whether Bax-mediated apoptosis is instrumental to the development of retinal microangiopathy, and, if so, provide proof of concept that apoptosis is a key cellular event in the development of diabetic retinopathy; (4) examine the retinal expression of TNF-alpha and other cytokines in human and experimental diabetes, and, if warranted, determine--by studying mice with targeted deletion of both TNF-alpha receptors and rendered diabetic--, whether TNF-alpha effects are instrumental or contributory to the development of diabetic retinal microangiopathy. These studies bridge biochemical abnormalities caused by hyperglycemia to altered regulation of apoptogenic molecules, and the latter to the histological lesions that render retinopathy a sight-threatening complication of diabetes. If a chain of causal relationships is established, the studies will bring to the fore targets for rational interventions.